生物技术通报 ›› 2022, Vol. 38 ›› Issue (5): 84-92.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1305

• 研究报告 • 上一篇    下一篇

2-十三烷酮胁迫下棉铃虫FoxAl调控CYP6B6的表达

魏倩1(), 刘小宁1(), 赵洁2()   

  1. 1.新疆大学生命科学与技术学院,乌鲁木齐 830046
    2.石河子大学农学院,石河子 832003
  • 收稿日期:2021-10-15 出版日期:2022-05-26 发布日期:2022-06-10
  • 作者简介:魏倩,女,硕士研究生;研究方向:昆虫分子生物学;E-mail: weiqian4130@163.com
  • 基金资助:
    国家自然科学基金项目(32001913);国家自然科学基金项目(31972279)

FoxAl Regulating CYP6B6 Expression Under 2-tridecanone Stress in Helicoverpa armigera

WEI Qian1(), LIU Xiao-ning1(), ZHAO Jie2()   

  1. 1. College of Life Science and Technology,Xinjiang University,Urumqi 830046
    2. Agriculture College,Shihezi University,Shihezi 832003
  • Received:2021-10-15 Published:2022-05-26 Online:2022-06-10

摘要:

旨在研究2-十三烷酮胁迫对棉铃虫Helicoverpa armigera叉头框A类似蛋白(forkhead box A-like protein,FoxAl)基因表达水平的影响,及FoxAl蛋白是如何调控解毒酶基因CYP6B6表达,为进一步明确FoxAl参与棉铃虫解毒代谢和生长发育过程提供依据。通过酵母自激活检测FoxAl蛋白的转录激活能力;通过凝胶阻滞检测FoxAl蛋白与CYP6B6启动子的结合能力;通过RNAi沉默棉铃虫5龄幼虫中肠内FoxAl基因,检测不同时间(24,48,72和 96 h)后FoxAlCYP6B6的表达变化情况;最后通过qPCR检测不同浓度(5,10和20 mg/g)2-十三烷酮处理不同时间(6,12,20,30和48 h)后棉铃虫6龄幼虫中肠内FoxAlCYP6B6的表达谱,并分析FoxAlCYP6B6表达谱的相关性。FoxAl蛋白具有激活酵母MEL1报告基因转录的能力,且该蛋白能与CYP6B6启动子中响应植物次生物质应答的核心片段结合。利用dsFoxAl沉默棉铃虫5龄幼虫中肠内FoxAl的表达量后,CYP6B6的表达量也显著降低。2-十三烷酮处理棉铃虫6龄幼虫后,中肠内FoxAlCYP6B6的表达量变化情况相似,基本都呈抛物线趋势,在短时间12 h内两者的表达量迅速上升后,随胁迫时间的延长两者的表达量逐渐降低;此外,FoxAlCYP6B6的表达量变化基本呈正相关,甚至在20 mg/g的胁迫浓度以及12 h和48 h的胁迫时间下两者都是高度正相关(r=0.819,P=0.045;r=0.987,P=0.007;r=0.978,P=0.011)。棉铃虫FoxAl蛋白可能是CYP6B6的转录激活因子,在植物次生物质2-十三烷酮短期胁迫下,FoxAl的表达量增加,进一步上调CYP6B6的表达,从而参与棉铃虫对2-十三烷酮的解毒作用。

关键词: 棉铃虫, 叉头框类似蛋白, 细胞色素P450酶, 2-十三烷酮, 转录因子

Abstract:

This study aims to investigate the effect of 2-tridecaneone stress on the gene expression of forkhead box A-like protein(FoxAl)in Helicoverpa armigera,and how FoxAl protein regulates the expression of detoxification enzyme gene CYP6B6,so as to provide a basis for further clarifying the role of FoxAl in detoxification metabolism and growth and development of H. armigera. Firstly,yeast self-activation test was applied to detect the transcriptional activation ability of FoxAl protein,and electrophoretic mobility shift assay was used to detect the binding ability of FoxAl protein to CYP6B6 promoter. Expression changes of FoxAl and CYP6B6 after different times(24,48,72 and 96 h)were detected after silencing FoxAl gene in the midgut of 5th H. armigera instar larva by RNAi. Finally qPCR was used to detect the expression profiles of FoxAl and CYP6B6 in the midgut of different 2-tridecone concentrations(5,10 and 20 mg/g)treated 6th instar larvae of H. armigera at different time post treatment(6,12,20,30 and 48 h),and their Pearson correlation was analyzed. FoxAl protein had the ability to activate transcription of MEL1 reporter gene in yeast,and it bound to the core fragment of CYP6B6 promoter in response to plant secondary substances. Using dsFoxAl to silence FoxAlexpression,the expression of CYP6B6 also significantly reduced in the midgut of 5th instar larvae of H. armigera. After the 6th instar larvae of H. armigera were treated with 2-tridecanone,the expressions of FoxAl and CYP6B6 in the midgut had similarly changed,basically showing a parabolic trend. The expressions of both rapidly increased within 12 h,and then gradually decreased with the extension of stress time. After with 2-tridecaneone treated,the expressions of FoxAl and CYP6B6 were basically positive correlation. They were highly positive correlation coefficients(r=0.819,P=0.045;r=0.987,P=0.007;r=0.978,P=0.011)even at the stress concentration of 20 mg/g and the stress times of 12 and 48 h. In conclusion,FoxAl protein may be a transcriptional activator of CYP6B6 in H. armigera. Under the short-term stress of plant secondary substance such as 2-tridecone,the expression of FoxAl increased,further up-regulated the expression of CYP6B6,thus participating in the detoxification to 2-tridecone in H. armigera.

Key words: Helicoverpa armigera, forkhead box like protein, cytochrome P450 enzyme, 2-tridecanone, transcriptional factor